Abstract This mentored career development award will support an early-career scientist studying human gastric stem cell (GSC) function in the context of Wnt signaling and the underlying mechanisms that give rise to dysplastic fundic gland polyps and antral adenomas in Familial Adenomatous Polyposis (FAP). The goals of this proposal are to understand how the Wnt signaling pathway regulates human gastric stem cell homeostasis, and to translate the basic biology of Wnt signaling to elucidate how pre-neoplastic gastric polyps develop in patients with FAP. Current cancer theory suggests that adult gastrointestinal (GI) stem cells that normally maintain tissue homeostasis can accumulate mutations that sustain tumor growth. My preliminary studies suggest that in the adult mouse stomach, Wnt pathway activity occurs in the proliferative zone of the stomach, as well as in stromal cells that surround stem and progenitor cells, suggesting a role for Wnt in supporting adult gastric stem cell homeostasis. This is also supported by the common occurrence of hyperplastic gastric polyps in FAP patients that carry Wnt activating mutations. However, the molecular mechanism for polyp development in these patients is unknown. I have recently established the 3D human organoid culture system from control gastric tissues, gastric polyps and gastric adenocarcinoma samples. The overarching hypothesis for this proposal is that adult GSC maintenance requires the Wnt signaling pathway, and Wnt pathway activation induces stem cell hyperproliferation necessary to initiate gastric pre-neoplasia. Aim 1 will elucidate the mechanism by which Wnt signaling supports stem cell homeostasis in the mammalian stomach through pharmacologic manipulation of Wnt signaling in human gastric organoids established from control and FAP gastric polyp tissues. Additionally, gene expression profiling will be used to identify the Wnt ligands and receptors that are expressed in the human adult gastric epithelium. Complimentary studies in mouse genetic models will be performed. I will also use Wnt reporter mouse models to identify Wnt-active gastric cell types. In Aim 2, I will use pathway-specific gene arrays to test Wnt-regulated genes such as stem cell markers and tumor suppressor genes. I will also use CRISPR/Cas9 gene editing technology to mutate TP53 in control and FAP human gastric organoids and test tumorigenicity in xenograft models. These experiments will establish whether mutations in tumor suppressor genes such as TP53 can promote neoplastic progression of FAP gastric polyps. Overall, these studies will provide the first in-depth analysis of Wnt regulation of human GSC function during normal homeostasis and FAP gastric polyp development. I expect my findings to inform future therapies for treatment of pre-neoplastic gastric disease.